The Golgi apparatus has two primary functions: biosynthesis of glycoproteins and glycolipids, and sorting. These functions underlie the elemental architecture of eukaryotic cells, so understanding how the Golgi functions is of fundamentally important. In addition, many human diseases (muscular deficiencies, lysosomal storage diseases, degenerative disease, etc) result from deficiencies of Golgi function. The ordered addition of carbohydrate moieties to biosynthetic cargo in the Golgi is carried out by glycosyltransferases that function sequentially, such that the products of early acting enzymes are substrates for later-acting enzymes. The location of enzymes in the Golgi stack parallels the glycosylation reactions;early- acting glycosyltransferases are enriched in cis/medial Golgi compartments while later-acting enzymes are enriched in medial/trans compartments. How is secretory cargo distinguished from Golgi residents so that cargo moves anterograde through the Golgi while Golgi residents are retained? Using budding yeast (Saccharomyces cerevisiae) to investigate sorting reactions in the Golgi, we discovered that a cytosolic protein, Vps74, directly recognizes the cytosolic portions of a subset of Golgi mannosyltransferases and is required to retain them in the Golgi. We hypothesize that Vps74 sorts Golgi residents into the retrograde pathway, however, the human ortholog of Vps74, GOLPH3, is reported in the literature to promote anterograde secretory transport from the Golgi. Preliminary data show that recruitment of GOLPH3 and Vps74 to the cytosolic leaflets of Golgi membranes requires ongoing synthesis of PtdIns4P, a phosphoinositide that is enriched in Golgi membranes and is required for both anterograde and retrograde Golgi trafficking. Using X-ray crystallography and lipid binding assays, we have identified a candidate PtdIns4P binding site on GOLPH3 and Vps74 and will elucidate a structure of GOLPH3/Vps74 in complex with PtdIns4P. Preliminary data also show that in a yeast vps74 mutant, PtdIns4P metabolism is altered, leading us to hypothesize that Vps74 and GOLPH3 regulate the production and/or turnover of 4-phosphorylated phosphoinositides. Altered phosphoinositide signaling at the Golgi is postulated to underlie the sorting defects that result from a loss of Vps74 and GOLPH3 function. GOLPH3 has recently been identified as a candidate oncogene that results in transformation when overexpressed. By combining functional studies in yeast and cultured human cells with biochemical, biophysical, and structural analyses of Vps74 and GOLPH3, these studies will resolve fundamental roles of PtdIns4P regulation in the Golgi, and elucidate the function of GOLPH3, which will shed light on its role in both normal and disease states. PUBLIC HEALTH RELEVANCE: In order for secreted proteins (hormones, nutrient and ion transporters, signaling receptors, etc) and lipids to function properly, they must be sequentially modified in the Golgi apparatus by enzymes that attach chains of sugar molecules to them. This research project investigates the structure and functions of proteins that function to assemble and maintain the organization of Golgi enzymes. One of these proteins, GOLPH3, has been implicated in causing a wide variety of cancers, so knowledge gained from this project will provide basic information regarding the assembly of the Golgi apparatus and help elucidate how GOLPH3 causes cancer.